Figure 2. Gel of pNIC-Bsa4 after restriction enzyme digest and PCR clean up.
Figure 1. Nanodrop of pNIC-Bsa4 after restriction enzyme digest and PCR clean up
Analysis: Concentration at 260nm wavelength had an absorbance of 111.5 ng/uL
120042014- Good work, but don't forget a conclusion next time
Week 11, 12, and 13
PCR cleanup
Figure 2. Nano drop image of PCR clean up product concentration.
Analysis: Maximum absorbance was at 260nm which confirms that sample contains DNA. The concentration at maximum absorbance was of 130.3 ng/uL, which is a good concentration. The next step is to move on to cloning.
PCR squared
Figure 1. Image of PCR squared gel. From left to right, first lane was skipped. This was followed by 1kb DNA ladder. The next four lanes are DNA products solutions from PCR squared.
Analysis: The PCR squared protocol followed was the same as secondary PCR (NEB guidelines) except for the increase from 20 cycles to 30 cycles. The bands are visible in the gel at the right length. Since PCR squared was successful and there didn't seem to be any major errors, the next step was to move on to PCR clean up.
1162014- Where is week 9,10? good job with week 7&8
9232014- You should elaborate on your analysis
Week 9 and 10
Secondary PCR
Figure 1. Secondary PCR 3rd trial solution is in the lane to the right of DNA 1kb lane. The DNA 1kb lane is in the second lane after the skipped lane.
Analysis: There is a visible band seen were the secondary PCR solution was run. The band is light, however this could have occurred because the ethidium bromide was not mixed correctly, which could have also caused to white spots at the top. The next step is to run PCR squared which will also confirm that secondary PCR worked.
Week 7 and 8
Secondary PCR 1st and 2nd Trial Re-run
Figure 1. Image of gel running secondary PCR, 1st and 2nd trail running in lane 4 and 5, respectively. Lane 2 contains DNA 1kb ladder.
Analysis: Lane five has a barely visible smudge, however, since it is not clear enough, the secondary PCR will be run again. This gel demonstrated that the error was not in the gel, and was in the conditions in which the secondary PCR was run. Therefore the times at which the PCR was run will be changed in the next run to the NEB recommended guidelines.
Secondary PCR 2nd Trial
Figure 1. Second trial for secondary PCR gel. DNA 1kb ladders at both lanes at the end of the gel. In lane 7 from left to right contains the solution from secondary PCR.
Analysis: Second trial for secondary PCR was run under the same conditions as the first trial and gave the same results of showing no bands or even a smudge. Errors could have occurred from making the mixture incorrectly or the process of running the gel could have also caused error. To test if the gel was not run incorrectly the first and second trial of secondary PCR will be run again.
Week 5 and 6
Secondary PCR
Figure 1. Gel for secondary PCR solution. The first lane after skipping a lane contains 1kb DNA ladder. Six lanes to the right of the ladder is the solution from secondary PCR.
Analysis: There was clear band shown in the gel from secondary PCR, therefore PCR did give the expected results. Secondary PCR will be ran one more time in the same conditions.
Primary PCR
Figure 1. Gel of primary PCR, first trial. Solution from primary PCR is in the lane to the right of 1kb DNA ladder on the left
Figure 2. Gel of the second trial for primary PCR. Solution of second trial primary PCR is to the right to 1kb DNA ladder that is further to the right. The lane to the right of lane with solution of second trial PCR is the solution for the first trial of primary PCR.
Analysis: The first gel of primary PCR showed that the PCR procedure did not work. However, when the second gel was ran, it showed that primary PCR had worked on the first trial and not on the second trial.
Plasmid Midi-Prep
Figure 1. Nanodrop trial 1 for pNIC concentration for MidiPrep
Figure 2. Nanodrop trial 2 for pNIC concentration for MidiPrep
Analysis: The average concentration for the two nano drop trails the average concentration was 59.3 ng/ul at the 260 wavelength. This concentration is in the range for what the range should be for pNIC, 30-60.
09232014- Keep up the good work, don't forget to include your captions
Week 3 and 4
PCR
Figure 1. First trial of nanodrop absorbance measurement for PCR
Figure 2. Second trial of nanodrop absorbance measurement for PCR
Figure 3. Agarose gel of PCR products. One lane was skipped, the first lane contains 5ul of ladder 100bp. Lane 2 contains a 1:10,000 template dilution, lane 3 contains a 1:1,000 template dilution, and lane 4 contains 1:100 template dilution. The fifth lane has no DNA template added.
Analysis: This PCR did not turn out properly. As it can be seen from the gel only the ladder came out correctly. The last lane contains DNA which should have been the one without DNA since it is the control. Lane 2, 3, and 4 don't contain any DNA which should have DNA.
Restriction Enzyme Digest
Figure 1. Agarose gel for RE digest. One lane was skipped. First lane contains 1kbs DNA ladder. Second lane contains uncut plasmid. Lane three contains plasmid and RE EcoRI. Lane 4 contains plasmid and RE enzyme PVUII. Lane 5 contains plasmid and both restriction enzymes.
Analysis: The bands did not appear as expected. The ladder did appear correctly and the uncut plasmid lane also appeared. However, the lanes that contains the restriction enzymes did not contain plasmid.
Day two and three ofBacterial Transformationof pNIC-Bsa4 E. coli DH-5 alpha cells
Figure 1. 500ml Erlenmeyer flask containing 80mL of LB+50ug/ml of Kanamycin with plasmid pNIC-Bsa4 in DH5alpha
Figure 2. 50mL conical tubes containing pellet of DH5 alpha with pNIC-Bsa4
Analysis: The pellets showed that the transformation was successful. The plasmid was taken by the bacteria and the pellet was able to be separated from the liquid.
Week 1 and 2
Bacterial Transformationof pNIC-Bsa4 E. coli DH-5 alpha cells
Figure 1. Agar plate containing cultures of 10ul DH5alpha E. coli with DNA plasmid pNIC-Bsa4 after 1 day of incubation
Figure 2. Agar plate containing cultures of 50ul DH5alpha E. coli with DNA plasmid pNIC-Bsa4 after 1 day of incubation
Analysis:
From the results above it can be seen that the bacteria did grow in one day of incubation. It can be observed that more bacteria cultures grew in the agar plate containing 50ul than the agar plate with 10ul of bacteria. The next step would be to grow the bacteria and then isolate the desired protein created by the bacteria.
Nanodrop
Figure 1. First trial of nanodrop absorbance measurement of pGBR22 plasmid
Figure 2. Second trial of nanodrop absorbance measurement of pGBR22 plasmid
Analysis:
The results demonstrated that the maximum absorbance was at the 260nm wavelength. This indicates that the nanodrop solution does in fact contain the DNA, since most DNA absorbs at the 260nm wavelength. After determining the average concentration, which was 241.3 ng/ul, the solution can be prepared and sent to DNA sequencing to confirm that the plasmid is pGBR22.
Week 14 & 15
Preparation of pNIC-Bsa4 as Accepting Vector
Analysis: Concentration at 260nm wavelength had an absorbance of 111.5 ng/uL
120042014- Good work, but don't forget a conclusion next time
Week 11, 12, and 13
PCR cleanup
Analysis: Maximum absorbance was at 260nm which confirms that sample contains DNA. The concentration at maximum absorbance was of 130.3 ng/uL, which is a good concentration. The next step is to move on to cloning.
PCR squared
Analysis: The PCR squared protocol followed was the same as secondary PCR (NEB guidelines) except for the increase from 20 cycles to 30 cycles. The bands are visible in the gel at the right length. Since PCR squared was successful and there didn't seem to be any major errors, the next step was to move on to PCR clean up.
1162014- Where is week 9,10? good job with week 7&8
9232014- You should elaborate on your analysis
Week 9 and 10
Secondary PCR
Analysis: There is a visible band seen were the secondary PCR solution was run. The band is light, however this could have occurred because the ethidium bromide was not mixed correctly, which could have also caused to white spots at the top. The next step is to run PCR squared which will also confirm that secondary PCR worked.
Week 7 and 8
Secondary PCR 1st and 2nd Trial Re-run
Analysis: Lane five has a barely visible smudge, however, since it is not clear enough, the secondary PCR will be run again. This gel demonstrated that the error was not in the gel, and was in the conditions in which the secondary PCR was run. Therefore the times at which the PCR was run will be changed in the next run to the NEB recommended guidelines.
Secondary PCR 2nd Trial
Analysis: Second trial for secondary PCR was run under the same conditions as the first trial and gave the same results of showing no bands or even a smudge. Errors could have occurred from making the mixture incorrectly or the process of running the gel could have also caused error. To test if the gel was not run incorrectly the first and second trial of secondary PCR will be run again.
Week 5 and 6
Secondary PCR
Analysis: There was clear band shown in the gel from secondary PCR, therefore PCR did give the expected results. Secondary PCR will be ran one more time in the same conditions.
Primary PCR
Analysis: The first gel of primary PCR showed that the PCR procedure did not work. However, when the second gel was ran, it showed that primary PCR had worked on the first trial and not on the second trial.
Plasmid Midi-Prep
Analysis: The average concentration for the two nano drop trails the average concentration was 59.3 ng/ul at the 260 wavelength. This concentration is in the range for what the range should be for pNIC, 30-60.
09232014- Keep up the good work, don't forget to include your captions
Week 3 and 4
PCR
Analysis: This PCR did not turn out properly. As it can be seen from the gel only the ladder came out correctly. The last lane contains DNA which should have been the one without DNA since it is the control. Lane 2, 3, and 4 don't contain any DNA which should have DNA.
Restriction Enzyme Digest
Analysis: The bands did not appear as expected. The ladder did appear correctly and the uncut plasmid lane also appeared. However, the lanes that contains the restriction enzymes did not contain plasmid.
Day two and three of Bacterial Transformation of pNIC-Bsa4 E. coli DH-5 alpha cells
Analysis: The pellets showed that the transformation was successful. The plasmid was taken by the bacteria and the pellet was able to be separated from the liquid.
Week 1 and 2
Bacterial Transformation of pNIC-Bsa4 E. coli DH-5 alpha cells
Analysis:
From the results above it can be seen that the bacteria did grow in one day of incubation. It can be observed that more bacteria cultures grew in the agar plate containing 50ul than the agar plate with 10ul of bacteria. The next step would be to grow the bacteria and then isolate the desired protein created by the bacteria.
Nanodrop
Figure 1. First trial of nanodrop absorbance measurement of pGBR22 plasmid
Figure 2. Second trial of nanodrop absorbance measurement of pGBR22 plasmid
Analysis:
The results demonstrated that the maximum absorbance was at the 260nm wavelength. This indicates that the nanodrop solution does in fact contain the DNA, since most DNA absorbs at the 260nm wavelength. After determining the average concentration, which was 241.3 ng/ul, the solution can be prepared and sent to DNA sequencing to confirm that the plasmid is pGBR22.